Characterization of mesenchymal stem cells from bone marrow of

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CEDEME - Center of Development
of Experimental Models for
Medicine and Biology
Regeneration of brain tissue by mesenchymal stem cells in a
stroke model involves both raised nitric oxide and VEGF as
decreased oxidative stress and apoptosis to healthful levels
Prof. Clélia Rejane Antônio Bertoncini
Coordinator of the Laboratories of Genetic Control and Oxidative Stress
at CEDEME - Federal University of Sao Paulo - Brazil
• Stroke or Cerebral Vascular Accident
entails:
• 10% of total deaths / World
• Interruption in blood flow: Ischemic or hemorrhagic
• Causes: hypertension, hemorrhage,
blood vessel malformation, thrombus,
tumor, inflammation and injuries
• Importance of stem cell therapy for stroke
is related to:
• Differentiation potential;
• Production of bioactive substances and secretion of factors;
• Inexistence of rejection.
Initial challenges:
- Finding an appropriate model for adult stem cells therapy:
Stroke Prone Spontaneously Hypertensive Rat (SHRSP)
- Defining parameters to be measured: oxidative stress,
apoptosis, neurogenesis, morphology and secreted factors
- Finding methods to track the location of stem cells in vivo:
labeling with the inside cell fluorescent dye CFSE *
- To propose a cellular therapy for spontaneous stroke:
mesenchymal SC / neural cell inducers
* CFSE – carboxyfluoroscein succinimidyl ester
Stroke model: SPSHR
• SHRSP - Stroke Prone Spontaneously Hypertensive Rat
• Severe hipertension, blood pressure ~ 220 mm Hg , stroke ~ one year of age;
• Origin: Wistar
Wistar Kyoto (WK)
SHR
SPSHR
• Genetic errors in enzymes, coenzymes and cofactors that regulate blood pressure
• Deficient in tetrahydrobiopterin - BH4 - cofactor of NO synthases;
• Increased levels of superoxide in aorta, DNA damage-type 8-hydroxy guanosine,
apoptotic neuronal death, lesions in the cortex (CA1 and hippocampus)
• SOD in CA1 - CA3 and hippocampus, NOS in cortex (Kimoto-Kinoshita, 1999);
• They are also models of genetic hypertension and elevated oxidative stress.
Animal models for cell therapy of stroke:
Control: WKY
For injection of SC via cisterna magna: SHRSP
_____________________________________
N= 10/group, age = 3 months and 1 year
_____________________________________
Transplantation: Autologous or heterologous?
- Depending on the quality and viability of SC
Adult stem cells from bone marrow
Mesenchymal stem cells from bone marrow = 0,01 a 0,001%
Isolation of Mesenchymal Stem Cells (MSCs)
from Bone Marrow of Rats
ring with
MSCs
(A) Bone marrow perfusion with culture medium via femoral orifices
(B) Separation of cells by Ficoll gradient
Cultures of stem cells from the bone marrow of rats
A
B
Morphology of MSC. Bone marrow MSC exhibited fibroblast-like morphology. Images were
taken from cultures at 7 (A) and 14 (B) days after plating. (A) Note the presence of two types of
cells: small, round cells (hematopoietic stem cells) and long-shaped cells (MSC). Scale = 50 µm.
(B) At this stage, the cells displayed the typical “fibroblast-like” morphology shown here. The cells
did not spontaneously differentiate during culture expansion. Scale bar = 100µm.
MSC characterization. Fluorescent activated cell sorter of cell-surface antigen
expression The expanded attached, mesenchymal cells were positive for CD90 and
CD105, which are markers of MSC. In contrast, the cells were negative for other
markers of the hematopoietic lineage, including CD45, Cd11b, c-kit and Sca-1.
Characterization of mesenchymal stem cells from bone
marrow of rats by confocal microscopy - negative marking
cells were
negative for
markers of the
hematopoietic
lineage,
including CD45,
Cd11b, c-kit
and Sca-1
Characterization of mesenchymal stem cells from bone
marrow of rats by confocal microscopy: positive staining
MSCs were double stained in blue with DAPI nuclear stain CD90,
CD105 antigens. The bright red cells indicate positivity.
Scale bar = 20 μm.
Viability of stem cells from normotensive WKY
and hypertensive SHRSP by MTT *** p < 0,0001.
Quantitative analysis of the expression of bax and bcl-2 in mesenchymal cells
culture with normal and hypertensive animals at 3 or 12 months of age
Real Time RT-PCR markers of apoptosis markers in cultured mesenchymal
stem cells from WKY e SPSHR. * = p < 0,05; ** = p < 0,01. N = 10 /group.
- Bax and Bcl2 – heterodimers?
- Stem cells from young animals are more viable and less
susceptible to apoptosis when derived from WKY.
- Transplantation from WKY 3 months to SHRSP 1 year old.
Transplantation of mesenchymal stem cells for the brains of
SHRSP animals via cisterna magna
CFSE
Dapi
superposition
Photomicrograph of brain from control animals not treated with MSC - the hippocampus.
Only PBS was injected - No unspecific fluorescence
Superposition
7 days
15 days
30 days
Reduction of oxidative stress in the brain after stem cells transplantation
Lipid peroxidation
Confocal microscopy: Increased superoxide in
the brain isolated from stroke-prone
spontaneuosly hypertensive rats, compared
with their normotensive controls Wistar Kyoto,
as detected by Dihydroethidium, DHE – red
spots and DAPI (all blue nucleus). The MSC
migrated to places of greater production of
superoxide anion. Note the co-localization of
MSC (green) and superoxide anion (red) in
treated animals. Scale bar = 20µm. (B)
Quantification and analysis of figure pixilation.
Results are expressed as mean ± SEM,
represented by error bars; **p < 0.01; ***p <
0.001.
Redox regeneration to normal levels
Reduction of apoptosis: bax/bcl-2 ~ normal after MSCs therapy
Analysis of gene expression of bax and bcl-2 in brain tissue of WKY and SHRSP in different ages, before and after
MSC treatment. (A) Representative ethidium bromide electrophoresis gel of RT-PCR: (B) qRT-PCR gene expression
of bax. (C) qRT-PCR gene expression of bcl-2. Note that bax expression increases with age, whereas bcl-2 decreases, in
hypertensive rats but not in normotensive rats. Despite The MSC does not decrease the expression of bax, bcl-2
expression was increased in treated animals.; *p < 0.05; **p < 0.01; ***p < 0.001.
Expression of CD90,
a marker of
iundifferentiated
cells, in SHRSP rat
brain one month
after MSC
transplantation
Photomicrograph of animal brains treated with MSCs. Mesenchymal cells labeled with CFSE
(green) and injected into the cisterna magna did not differentiate into neurons, at least thirty days
after injection and showed labeling for CD90 (red). The nuclei are stained blue with DAPI.
Regeneration of hippocampal injury site in brain of Stroke
prone animals after stem cells transplantation
Photomicrographs of coronal sections of rat brains at the level of the hippocampus; dentate gyrus
stained by Nissl technique (cresyl). We note that there is no damage and disarray of cell layers in the
in treated animals. A and B, 40x magnification. C and D, 100x magnification.
Is Nitric oxide involved in brain damage regeneration?
Significant elevation of nitric oxide (NO) in brain of stroke prone rats, 30 days after
Stem cells transplantation. n = 10 / group. Values are means ± SE. *p < 0.01
O2 . + NO
ONOO-
MSCs from healthy animals can secrete and / or donate nitric oxide
to inactivate superoxide in endogenous brain cells of stroke model.
Is the proliferative factor Vegf involved
in brain damage regeneration?
Conclusions:
- Mesenchymal stem cells (MSCs) from stroke prone and
hypertensive animals have lower viability when compared with
normotensive animals;
- When injected into the brains of hypertensive animal model of
stroke, MSCs decreased neuronal death by reducing apoptotic
rate of these cells, mainly due to the reestablishment of Bcl-2;
- MSCs reduced oxidative stress to healthy levels in brain of
SHRSP, which indicates a potential antioxidant activity of these
cells;
-MSCs were able to reestablish the rearrangement of cell
layers in the hippocampus region of stroke prone animals;
- Nitric oxide and Vegf - secreted or induced by transplanted
MSCs - are factors involved in brain damage regeneration.
Free Radical Biology and Medicine 70 (2014), 141–154
Transplantation of bone marrow mesenchymal stem cells decreases
oxidative stress, apoptosis, and hippocampal damage in brain of a
spontaneous stroke model.
E-mails : michele.longoni@unifesp.br, clelia.rejane@unifesp.br
Group:
Clélia R. A. Bertoncini
Aline de Cassia Azevedo
Darci Souza Marinho
Colaborators:
Alice T. Ferreira (Biophysic)
Gui Mi Ko
Michele Longonni Calió
Milene Subtil Ormanji
Tatiana Pinotti Guirao
Telma Lisboa do Nascimento (UFRJ)
Luciana Reis (Nefrology)
Manuel de Jesus Simões (Morfology)
Elisa Higa (Nefrology)
Adriana Carbonel
Soraya Smaili (Pharmacology –UNIFESP)
Tatiana Rosenstock
Apoio: FAPESP, CNPq e UNIFESP
Laboratórios de Controle Genético e Estresse Oxidativo
CEDEME - Centro de Desenvolvimento de Modelos Experimentais para Medicina e Biologia
2010
e-mail: clelia.rejane@unifesp.br
2014
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